BmATG5 and BmATG6 mediate apoptosis following autophagy induced by 20-hydroxyecdysone or starvation

Autophagy and apoptosis, which could be induced by common stimuli, play crucial roles in development and disease. The functional relationship between autophagy and apoptosis is complex, due to the dual effects of autophagy. In the Bombyx Bm-12 cells, 20-hydroxyecdysone (20E) treatment or starvation-induced cell death, with autophagy preceding apoptosis. In response to 20E or starvation, BmATG8 was rapidly cleaved and conjugated with PE to form BmATG8–PE; subsequently, BmATG5 and BmATG6 were cleaved into BmATG5-tN and BmATG6-C, respectively. Reduction of expression of BmAtg5 or BmAtg6 by RNAi decreased the proportion of cells undergoing both autophagy and apoptosis after 20E treatment or starvation. Overexpression of BmAtg5 or BmAtg6 induced autophagy but not apoptosis in the absence of the stimuli, but promoted both autophagy and apoptosis induced by 20E or starvation. Notably, overexpression of cleavage site-deleted BmAtg5 or BmAtg6 increased autophagy but not apoptosis induced by 20E or starvation, whereas overexpression of BmAtg5-tN and BmAtg6-C was able to directly trigger apoptosis or promote the induced apoptosis. In conclusion, being cleaved into BmATG5-tN and BmATG6-C, BmATG5 and BmATG6 mediate apoptosis following autophagy induced by 20E or starvation in Bombyx Bm-12 cells, reflecting that autophagy precedes apoptosis in the midgut during Bombyx metamorphosis.     

Functional identification of Bombyx mori Atg13 in autophagy

The autophagy process involves a series of autophagy-related (Atg) proteins, which are conserved in eukaryotes. ULK1/Atg1-ATG13/Atg13 is the core protein complex for autophagy initiation in response to nutrient and hormone signaling. However, how Atg13 is regulated to participate in autophagy is unclear in insects. Here in Bombyx mori, the variation of BmAtg13 was correlated with autophagy induced by steroid hormone 20-hydroxyecdysone (20E) or starvation. Developmental profiles from feeding to prepupal stage revealed that there were two bands of BmAtg13 protein detected by western blot analysis, therein the upper band was intensively decreased, while the lower band was significantly increased which was in accordance with its mRNA variation; and immunofluorescent staining indicated that BmAtg13 was nucleocytoplasmic translocated during larval-pupal metamorphosis when autophagy was dramatically induced. BmAtg13 knockdown and overexpression both inhibits autophagy. Besides, 20E treatment-induced BmAtg13 gene expression, while blocking 20E signaling transduction by knockdown of BmUsp reduced both gene expression and protein level of BmAtg13. These results reveal that BmAtg13 is required for 20E- and starvation-induced autophagy in B. mori, which provides the foundation for further related studies.     

BmCaspase-8-like regulates autophagy by suppressing BmDREDD-mediated cleavage of BmATG6

Autophagy plays an important role in tissue remodeling during insect development. The interplay between autophagy-related (ATG) proteins and caspases regulates the autophagic activity of ATGs, thereby modulating the process of autophagy. Our previous study characterized BmCaspase-8-like (BmCasp8L) as a caspase suppressor that inhibits apoptosis and immune signaling by suppressing the activation of death-related ced-3/Nedd2-like caspase (DREDD), a caspase-8 homolog in silkworm. In this study, we explored the regulatory role of BmCasp8L in autophagy. We found that the expression of Bmcasp8l increased from the late spinning stage to the pupa stage in the posterior silk gland (PSG), correlating with the expression patterns of Bmatg8 and Bmatg6. RNA interference-mediated downregulation of BmCasp8L expression significantly decreased starvation-induced autophagic influx as determined by the levels of BmATG8–phosphatidylethanolamine and the percentage of cells displaying punctate enhanced green fluorescent protein-BmATG8. Conversely, the overexpression of BmCasp8L significantly increased autophagic influx. We also found that BmCasp8L underwent autophagic degradation induced by starvation and that it was colocalized with BmATG8. Lastly, we demonstrated that BmDREDD attenuated autophagy and BmCasp8L suppressed BmDREDD-mediated cleavage of BmATG6. Taken together, our results demonstrated that BmCasp8L is a novel proautophagic molecule which suppresses BmDREDD-mediated cleavage of BmATG6 and is a target for autophagy.     

Calpain‐truncated CRMP‐3 and ‐4 contribute to potassium deprivation‐induced apoptosis of cerebellar granule neurons

Increasing evidence shows that calpain‐mediated proteolytic processing of a selective number of proteins plays an important role in neuronal apoptosis. Study of calpain‐mediated cleavage events and related functions may contribute to a better understanding of neuronal apoptosis and neurodegenerative diseases. We, therefore, investigated the role of calpain substrates in potassium deprivation‐induced apoptosis of cerebellar granule neurons (CGNs). Twelve previously known and seven novel candidates of calpain substrates were identified by 2‐D DIGE and MALDI‐TOF/TOF MS analysis. Further, the identified novel calpain substrates were validated by Western blot analysis. Moreover, we focused on the collapsin response mediator proteins (CRMP‐1, ‐2, ‐3 and ‐4 isoforms) and found that CRMPs were proteolytically processed by calpain but not by caspase, both in vivo and in vitro. To clarify the properties of the calpain‐mediated proteolysis of CRMPs, we constructed the deletion mutants of CRMPs for additional biochemical studies. In vitro cleavage assays revealed that CRMP‐1, ‐2 and ‐4 were truncated by calpain at the C‐terminus, whereas CRMP‐3 was cleaved at the N‐terminus. Finally, we assessed the role of CRMPs in the process of potassium deprivation‐triggered neuronal apoptosis by overexpressing the truncated CRMPs in CGNs. Our data clearly showed that the truncated CRMP‐3 and ‐4, but not CRMP‐1 and ‐2, significantly induced neuronal apoptosis. These findings demonstrated that calpain‐truncated CRMP‐3 and ‐4 act as pro‐apoptotic players when CGNs undergo apoptosis.     

Synthesis and Anti-juvenile Hormone Activity of 1-Citronellyl-5-substituted Imidazoles

A number of 1-citronellyl-5-substituted imidazoles were synthesized and bioassayed on the silkworm, Bombyx mori, in order to assess their anti-juvenile hormone activity. Most of the 1-citronellyl-5-substituted imidazoles induced precocious metamorphosis in the 3rd and 4th instar larvae of B. mori by topical application. The percentage of precocious metamorphosis correlated well with the dosage. Among the compounds tested, l-citronellyl-5-(2-chlorophenyl)imidazole (8) and the 2-methylphenyl analog 10 showed the highest activity. When compounds 8 and 10 were applied to the 4th instar larvae at 10 μg/larva, precocious pupation was induced in 100% without any lethal effects.     

Calpain‐2 plays a pivotal role in the inhibitory effects of propofol against TNF‐α‐induced autophagy in mouse hippocampal neurons

Calpains are calcium‐dependent proteases and play critical roles in neuronal autophagy induced by inflammation. Propofol has been reported to exert anti‐inflammatory effects in neurons. We aimed to identify whether and how propofol‐modulated calpain activity and neuron autophagy in response to tumour necrosis factor‐α (TNF‐α). Mouse hippocampal neurons were pre‐treated with propofol and exposed to TNF‐α. Autophagy was evaluated by fluorescent autophagy assay and by measuring LC3I and LC3II expression. Intracellular calcium concentration was measured by fluorescent assay. Calpain activation was measured by calpain activity assay. The protein expression of intracellular signalling molecules was detected by Western blot analysis. Compared with untreated control neurons, 40 ng/mL TNF‐α treatment for 2 hours induced neuron autophagy, which was attenuated by 25 μmol/L propofol. TNF‐α induced intracellular calcium accumulation, the phosphorylation of calcium/calmodulin‐dependent protein kinase II (CAMK II) and calpain‐2, calpain activation and lysosomal cathepsin B release as well as tyrosine kinase receptor B (TrkB) truncation. These effects were alleviated by propofol, calcium chelator, CAMK II inhibitor, calpain‐2 inhibitor, calpain‐2 siRNA transfection and N‐Methyl‐d ‐aspartic acid (NMDA) receptor antagonist. Propofol, via NMDA receptor, inhibited TNF‐α‐mediated hippocampal neuron autophagy. The mechanism may involve calcium and calcium‐dependent signalling pathway, especially CAMK II and calpain‐2.     

Drosophila Atg7: Required for stress resistance,longevity and neuronal homeostasis,but not for metamorphosis

Autophagy, the lysosomal degradation and recycling of self material, has been implicated in a number of developmental and pathological conditions including aging, cancer, neurodegeneration, and insect metamorphosis. Surprisingly, Atg7 mutant flies are able to complete metamorphosis with only a slight delay, despite strongly reduced autophagy levels. Similarly, developmental elimination of the larval midgut proceeds with normal morphology, suggesting that animals can compensate for reduced autophagy during development. Atg7 mutant adults are hypersensitive to starvation and oxidative stress, live shorter, and accumulate ubiquitin-positive aggregates in the brain that lead to a progressive decline of neuronal function and cell death. These results suggest that in Drosophila, normal levels of autophagy may play a more important role in the homeostasis of certain terminally differentiated cells and stress survival than during development.

Addendum to: Juhász G, Érdi B, Sass M, Neufeld TP. Atg7-dependent autophagy promotes neuronal health, stress tolerance, and longevity but is dispensable for metamorphosis in Drosophila. Genes Dev 2007; 21:3061-6.     

A diphenyldiselenide derivative induces autophagy via JNK in HTB‐54 lung cancer cells

Symmetric aromatic diselenides are potential anticancer agents with strong cytotoxic activity. In this study, the in vitro anticancer activities of a novel series of diarylseleno derivatives from the diphenyldiselenide (DPDS) scaffold were evaluated. Most of the compounds exhibited high efficacy for inducing cytotoxicity against different human cancer cell lines. DPDS 2 , the compound with the lowest mean GI₅₀ value, induced both caspase‐dependent apoptosis and arrest at the G₀/G₁ phase in acute lymphoblastic leucemia CCRF‐CEM cells. Consistent with this, PARP cleavage; enhanced caspase‐2, ‐3, ‐8 and ‐9 activity; reduced CDK4 expression and increased levels of p53 were detected in these cells upon DPDS 2 treatment. Mutated p53 expressed in CCRF‐CEM cells retains its transactivating activity. Therefore, increased levels of p21^CIP1 and BAX proteins were also detected. On the other hand, DPDS 6 , the compound with the highest selectivity index for cancer cells, resulted in G₂/M cell cycle arrest and caspase‐independent cell death in p53 deficient HTB‐54 lung cancer cells. Autophagy inhibitors 3‐methyladenine, wortmannin and chloroquine inhibited DPDS 6 ‐induced cell death. Consistent with autophagy, increased LC3‐II and decreased SQSTM1/p62 levels were detected in HTB‐54 cells in response to DPDS 6 . Induction of JNK phosphorylation and a reduction in phospho‐p38 MAPK were also detected. Moreover, the JNK inhibitor SP600125‐protected HTB‐54 cells from DPDS 6 ‐induced cell death indicating that JNK activation is involved in DPDS 6 ‐induced autophagy. These results highlight the anticancer effects of these derivatives and warrant future studies examining their clinical potential.     

Crystal structure of Bombyx mori arylphorins reveals a 3:3 heterohexamer with multiple papain cleavage sites

In holometabolous insects, the accumulation and utilization of storage proteins (SPs), including arylphorins and methionine‐rich proteins, are critical for the insect metamorphosis. SPs function as amino acids reserves, which are synthesized in fat body, secreted into the larval hemolymph and taken up by fat body shortly before pupation. However, the detailed molecular mechanisms of digestion and utilization of SPs during development are largely unknown. Here, we report the crystal structure of Bombyx mori arylphorins at 2.8 Å, which displays a heterohexameric structural arrangement formed by trimerization of dimers comprising two structural similar arylphorins. Our limited proteolysis assay and microarray data strongly suggest that papain‐like proteases are the major players for B. mori arylphorins digestion in vitro and in vivo. Consistent with the biochemical data, dozens of papain cleavage sites are mapped on the surface of the heterohexameric structure of B. mori arylphorins. Hence, our results provide the insightful information to understand the metamorphosis of holometabolous insects at molecular level.     

Expression profile of cathepsin B in the fat body of Bombyx mori during metamorphosis

Proteolytic enzymes are involved in insect molting and metamorphosis, and play a vital role in the programmed cell death of obsolete organs. Here we show the expression profile of cathepsin B in the fat body of the silkworm Bombyx mori during development. We also compare the expression profiles of B. mori cathepsins B (BmCatB) and D (BmCatD) during normal development and after RNA interference (RNAi)-mediated inhibition. BmCatB is induced by 20-OH-ecdysone, and is expressed in the fat body of B. mori during molting and the larval–pupal and pupal–adult transformations, where its expression leads to programmed cell death. In particular, BmCatB is highly expressed in the fat body of B. mori during the larval–pupal transformation, and BmCatB RNAi treatment resulted in an arrest of the larval–pupal transformation. RNAi-mediated BmCatB knockdown sustained the expression of BmCatD during the larval–pupal transformation. On the other hand, when BmCatD was inhibited via RNAi, the expression of BmCatB was upregulated. Based on these results, we conclude that BmCatB is involved in the programmed cell death of the fat body during B. mori metamorphosis, and that BmCatB and BmCatD contribute to B. mori metamorphosis.     

Starvation‐induced MTMR13 and RAB21 activity regulates VAMP8 to promote autophagosome–lysosome fusion

Autophagy, the process for recycling cytoplasm in the lysosome, depends on membrane trafficking. We previously identified Drosophila Sbf as a Rab21 guanine nucleotide exchange factor (GEF) that acts with Rab21 in endosomal trafficking. Here, we show that Sbf/MTMR13 and Rab21 have conserved functions required for starvation‐induced autophagy. Depletion of Sbf/MTMR13 or Rab21 blocked endolysosomal trafficking of VAMP8, a SNARE required for autophagosome–lysosome fusion. We show that starvation induces Sbf/MTMR13 GEF and RAB21 activity, as well as their induced binding to VAMP8 (or closest Drosophila homolog, Vamp7). MTMR13 is required for RAB21 activation, VAMP8 interaction and VAMP8 endolysosomal trafficking, defining a novel GEF‐Rab‐effector pathway. These results identify starvation‐responsive endosomal regulators and trafficking that tunes membrane demands with changing autophagy status.     

A new ferulic acid ester, a new ellagic acid derivative, and other constituents from pachycentria formosana: effects on neutrophil pro-inflammatory responses

A new ferulic acid ester derivative, tetracosane‐1,24‐diyl di[(Z)‐ferulate] ( 1 ), and a new ellagic acid derivative, 3,4 : 3′,4′‐bis(O,O‐methylene)ellagic acid ( 2 ), have been isolated from leaves and twigs of Pachycentria formosana, together with eight known compounds. Their structures were determined by in‐depth spectroscopic and mass‐spectrometric analyses. Among the isolated compounds, oleanolic acid ( 6 ), ursolic acid acetate ( 7 ), and 3‐epibetulinic acid ( 9 ) exhibited potent inhibition (IC₅₀ values ≤21.8 μM ) of O₂^⋅− generation by human neutrophils in response to N‐formyl‐L ‐methionyl‐L ‐leucyl‐L ‐phenylalanine/cytochalasin B (fMLP/CB). In addition, oleanolic acid ( 6 ), 3‐O‐[(E)‐feruloyl]ursolic acid ( 8 ), 3‐epibetulinic acid ( 9 ), and lawsonic acid ( 10 ) also inhibited fMLP/CB‐induced elastase release with IC₅₀ values ≤18.6 μM .     

Lipidation of BmAtg8 is required for autophagic degradation of p62 bodies containing ubiquitinated proteins in the silkworm,Bombyx mori

p62/Sequestosome-1 (p62/SQSTM1, hereafter referred to as p62) is a major adaptor that allows ubiquitinated proteins to be degraded by autophagy, and Atg8 homologs are required for p62-mediated autophagic degradation, but their relationship is still not understood in Lepidopteran insects. Here it is clearly demonstrated that the silkworm homolog of mammalian p62, Bombyx mori p62 (Bmp62), forms p62 bodies depending on its Phox and Bem1p (PB1) and ubiquitin-associated (UBA) domains. These two domains are associated with Bmp62 binding to ubiquitinated proteins to form the p62 bodies, and the UBA domain is essential for the binding, but Bmp62 still self-associates without the PB1 or UBA domain. The p62 bodies in Bombyx cells are enclosed by BmAtg9-containing membranes and degraded via autophagy. It is revealed that the interaction between the Bmp62 AIM motif and BmAtg8 is critical for the autophagic degradation of the p62 bodies. Intriguingly, we further demonstrate that lipidation of BmAtg8 is required for the Bmp62-mediated complete degradation of p62 bodies by autophagy. Our results should be useful in future studies of the autophagic mechanism in Lepidopteran insects.     

Calpain as a Novel Regulator of Autophagosome Formation

Ubiquitously expressed mu- and m-calpain proteases consist of 80-kDa catalytic subunits encoded by the Capn1 and Capn2 genes, respectively, and a common 28-kDa regulatory subunit encoded by the calpain small 1 (Capns1) gene.

The mu- and m-calpain proteases have been implicated in both pro- or anti-apoptotic functions. We have found that Capns1 depletion is coupled to increased sensitivity to apoptosis triggered by a number of autophagy-inducing stimuli in mammalian cells. Therefore we investigated the involvement of calpains in autophagy using MEFs derived from Capns1 knockout mice and Capns1 depleted human cells as model systems.

We found that autophagy is impaired in Capns1-deficient cells by immunostaining of the endogenous autophagosome marker LC3 and electron microscopy experiments. Accordingly, the enhancement of lysosomal activity and long-lived proteins degradation, normally occurring upon starvation, are also reduced.

In Capns1-depleted cells ectopic LC3 accumulates in early endosome-like vesicles that might represent a salvage pathway for protein degradation when autophagy is defective.

Addendum to:

Calpain is Required for Macroautophagy in Mammalian Cells

Francesca Demarchi, Cosetta Bertoli, Tamara Copetti, Isei Tanida, Claudio Brancolini, Eeva-Liisa Eskelinen and Claudio Schneider

J Cell Biol 2006; 175:595-605     

Overcoming resistance to mitochondrial apoptosis by BZML‐induced mitotic catastrophe is enhanced by inhibition of autophagy in A549/Taxol cells

Objectives

Our previous in vitro study showed that 5‐(3, 4, 5‐trimethoxybenzoyl)‐4‐methyl‐2‐(p‐tolyl) imidazol (BZML) is a novel colchicine binding site inhibitor with potent anti‐cancer activity against apoptosis resistance in A549/Taxol cells through mitotic catastrophe (MC). However, the mechanisms underlying apoptosis resistance in A549/Taxol cells remain unknown. To clarify these mechanisms, in the present study, we investigated the molecular mechanisms of apoptosis and autophagy, which are closely associated with MC in BZML‐treated A549 and A549/Taxol cells.

Methods

Xenograft NSCLC models induced by A549 and A549/Taxol cells were used to evaluate the efficacy of BZML in vivo. The activation of the mitochondrial apoptotic pathway was assessed using JC‐1 staining, Annexin V‐FITC/PI double‐staining, a caspase‐9 fluorescence metric assay kit and western blot. The different functional forms of autophagy were distinguished by determining the impact of autophagy inhibition on drug sensitivity.

Results

Our data showed that BZML also exhibited desirable anti‐cancer activity against drug‐resistant NSCLC in vivo. Moreover, BZML caused ROS generation and MMP loss followed by the release of cytochrome c from mitochondria to cytosol in both A549 and A549/Taxol cells. However, the ROS‐mediated apoptotic pathway involving the mitochondria that is induced by BZML was only fully activated in A549 cells but not in A549/Taxol cells. Importantly, we found that autophagy acted as a non‐protective type of autophagy during BZML‐induced apoptosis in A549 cells, whereas it acted as a type of cytoprotective autophagy against BZML‐induced MC in A549/Taxol cells.

Conclusions

Our data suggest that the anti‐apoptosis property of A549/Taxol cells originates from a defect in activation of the mitochondrial apoptotic pathway, and autophagy inhibitors can potentiate BZML‐induced MC to overcome resistance to mitochondrial apoptosis.

     

Molecular characterization of DnaJ 5 homologs in silkworm Bombyx mori and its expression during egg diapause

A comparison of the cDNA sequences （1 056 bp） of Bombyx mori DnaJ 5 homolog with B. mori genome revealed that unlike in other Hsps, it has an intron of 234 bp. The DnaJ 5 homolog contains 351 amino acids, of which 70 contain the conserved DnaJ domain at the N-terminal end. This homolog orB. mori has all desirable functional domains similar to other insects, and the 13 different DnaJ homologs identified in B. mori genome were distributed on different chromosomes. The expressed sequence tag database analysis of Hsp40 gene expression revealed higher expression in wing disc followed by diapause-induced eggs. Microarray analysis revealed higher expression of DnaJ 5 homolog at 18th h after oviposition in diapause-induced eggs. Further validation of DnaJ 5 expression through qPCR in diapause-induced and nondiapause eggs at different time intervals revealed higher expression in diapause eggs at 18 and 24 h after oviposition, which coincided with the expression of Hsp70 as the Hsp 40 is its co-chaperone. This study thus provides an outline of the genome organization of lisp40 gene, and its role in egg diapause induction in B. mori.